DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-induced Acute Kidney Injury in Mice Atsushi Iwamura, Katsuhito Watanabe, Sho Akai, Tsubasa Nishinosono, Koichi Tsuneyama, Shingo Oda, Toshiyuki Kume, and Tsuyoshi Yokoi Downloaded from DMPK Research Laboratory, Mitsubishi Tanabe Pharma Corporation, Saitama, Japan (A.I., T.K.); Department of Drug Safety Sciences, Nagoya University Graduate School dmd.aspetjournals.org of Medicine, Aichi, Japan (K.W., S.A., T.N., S.O., T.Y.); Department of Molecular and Environmental Pathology, Institute of Health Biosciences Tokushima University Graduate School, Tokushima, Japan (K.T), and Drug Metabolism and Toxicology, at ASPET Journals on September 30, 2021 Faculty of Pharmaceutical Sciences, Kanazawa University, Ishikawa, Japan (A.I.) 1 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 Running title: Kidney injury induced by zomepirac acyl glucuronide Address correspondence to: Atsushi Iwamura DMPK Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Downloaded from Kawagishi, Toda, Saitama 335-8505, Japan. Phone: +81-48-433-8419 dmd.aspetjournals.org Fax: +81-48-433-8170 E-mail: [email protected] at ASPET Journals on September 30, 2021 Number of text pages: 44 Number of tables: 1 Number of figures: 8 Number of references: 52 Number of words in the Abstract: 249 Number of words in the Introduction: 574 Number of words in the Discussion: 1471 ABBREVIATIONS: AG, acyl glucuronide; ALT, alanine aminotransferase; BUN, blood urea nitrogen; BSO, L-buthionine-(S,R)-sulfoximine; CRE, creatinine; GAPDH, 2 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; GSSG, disulfide glutathione; HO-1, heme oxygenase 1; HPLC, high-performance liquid chromatography; ICAM-1, intercellular adhesion molecule-1; IL-1α, interleukin 1 alpha; IL-6, interleukin 6; IL-8, interleukin 8; KPB, potassium phosphate buffer; MDA, malondialdehyde; MIP-2, macrophage inflammatory protein-2; PBMCs, peripheral Downloaded from blood mononuclear cells; S100A9, S100 calcium-binding protein A9; TBARS, thiobarbituric acid reactive substances; TOTP, tri-o-tolyl phosphate; UDPGA, dmd.aspetjournals.org UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase; ZP, zomepirac at ASPET Journals on September 30, 2021 3 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 Abstract Glucuronidation, an important phase II metabolic route, is generally considered to be a detoxification pathway. However, acyl glucuronides (AGs) have been implicated in the toxicity of carboxylic acid drugs due to their electrophilic reactivity. Zomepirac (ZP) Downloaded from was withdrawn from the market because of adverse effects such as renal toxicity. Although ZP is mainly metabolized to acyl glucuronide (ZP-AG) by dmd.aspetjournals.org UDP-glucuronosyltransferase, the role of ZP-AG in renal toxicity is unknown. In this study, we established a ZP-induced kidney injury mouse model by pretreatment with at ASPET Journals on September 30, 2021 tri-o-tolyl phosphate (TOTP), a non-selective esterase inhibitor, and L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor. The role of ZP-AG in renal toxicity was investigated using this model. The model showed significant increases in blood urea nitrogen (BUN) and creatinine (CRE), but not alanine aminotransferase. The ZP-AG concentrations were elevated by co-treatment with TOTP in the plasma and liver and especially in the kidney. The ZP-AG concentrations in the kidney correlated with values for BUN and CRE. Upon histopathological examination, vacuoles and infiltration of mononuclear cells were observed in the model mouse. In addition to immune-related responses, oxidative stress 4 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 markers such as the glutathione/disulfide glutathione ratio and malondialdehyde levels were different in the mouse model. The suppression of ZP-induced kidney injury by tempol, an antioxidant agent, suggested the involvement of oxidative stress in ZP-induced kidney injury. This is the first study to demonstrate that AG accumulation in the kidney by TOTP and BSO treatment could explain renal toxicity and to show the in Downloaded from vivo toxicological potential of AGs. dmd.aspetjournals.org at ASPET Journals on September 30, 2021 5 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 Introduction Acyl glucuronidation is one of the major metabolic routes of carboxylic acid-containing drugs. Glucuronidation is an important phase II metabolic pathway for endogenous and exogenous substrates and is generally considered as a detoxification Downloaded from pathway. However, acyl glucuronides (AGs) are unstable under physiological conditions and consequently undergo hydrolysis or intramolecular rearrangement through the dmd.aspetjournals.org migration of the drug moiety from the 1-O-position to the 2-, 3-, or 4-position on the glucuronic acid ring (Smith et al., 1990a; Benet et al., 1993; Bailey and Dickinson, at ASPET Journals on September 30, 2021 2003). Because of their electrophilic nature and ability to cause substitution reactions with nucleophilic groups in proteins or other macromolecules, AGs can covalently modify endogenous proteins and have been postulated to cause the adverse toxicity associated with carboxylic acid-containing drugs (Faed, 1984; Boelsterli, 2002). To assess the toxicity of AGs, several in vitro assay systems, such as stability assay by measuring half-lives in potassium phosphate buffer, peptide adducts assay and immunostimulation assay, have been proposed (Wang et al., 2004; Sawamura et al., 2010; Jinno et al., 2013; Miyashita et al., 2014; Iwamura et al., 2015). However, the toxicity of AGs has remained controversial because direct evidence of in vivo AGs 6 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 toxicity has not been provided. Zomepirac (ZP), a nonsteroidal anti-inflammatory drug, was withdrawn from the market because of adverse effects such as anaphylaxis and renal toxicity (Smith, 1982; Miller et al., 1983; Heintz, 1995). ZP is mainly metabolized to acyl glucuronide (ZP-AG) in humans (Grindel et al., 1980; O'Neill et al., 1982). ZP-AG is more Downloaded from physicochemically unstable in phosphate buffer than the AGs of safe drugs such as gemfibrozil, repaglunide and telmisartan (Sawamura et al., 2010). ZP-AG also dmd.aspetjournals.org covalently modifies dipeptidyl peptidase IV in rat liver homogenates and microtubular protein in the bovine brain in vitro (Bailey et al., 1998; Wang et al., 2001). We at ASPET Journals on September 30, 2021 previously demonstrated that ZP-AG showed the highest induction of the mRNA expression of immune- and inflammation-related genes in human peripheral blood mononuclear cells (PBMCs) in the AGs of 13 drugs (Iwamura et al., 2015). Although the toxicity of ZP-AG has been suggested, there is no evidence that ZP-AG is involved in ZP-induced toxicity in vivo in humans or laboratory animals because of the difficulty of toxicological assessment under the conditions required for sufficient exposure to ZP-AG in vivo. The level of AG production is determined by glucuronidation catalyzed by UGT and enzymatic hydrolysis. The enzymatic hydrolysis of AG is catalyzed by esterases 7 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 such as acylpeptide hydrolase and α/β hydrolase domain containing (ABHD) 10 (Suzuki et al., 2010; Iwamura et al., 2012). It was reported that the plasma clearance of ZP-AG in the guinea pig was decreased by phenylmethylsulfonyl fluoride, a general esterase inhibitor, suggesting that ZP-AG is hydrolyzed by esterases (Smith et al., 1990b). In other reports, esterases were potently inhibited by tri-o-tolyl phosphate (TOTP), a Downloaded from non-selective esterase inhibitor, in mice and rats in vivo (Silver and Murphy, 1981; Kobayashi et al., 2012). ZP-AG conjugates with glutathione (GSH) in rat hepatocytes dmd.aspetjournals.org and bile (Grillo and Hua, 2003). Therefore, the ZP-AG level is regulated via hydrolysis by esterases and GSH conjugation against the generation by UGT. It is assumed that the at ASPET Journals on September 30, 2021 increased exposure to ZP-AG in vivo by TOTP and L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor, after ZP administration may show that ZP-AG rather than ZP is involved in ZP-induced toxicity in vivo. The purpose of the present study was to establish the ZP-induced kidney injury mouse model and to investigate the role of ZP-AG in kidney injury. 8 DMD Fast Forward. Published on April 25, 2016 as DOI: 10.1124/dmd.116.069575 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 69575 Materials and Methods Chemicals and Reagents. Reduced GSH, oxidized GSH and BSO were purchased from Wako Pure Chemical Industries (Osaka, Japan). Zomepirac sodium and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) were obtained from Downloaded from Sigma-Aldrich (St.